Megakaryocytopoiesis studied in a mathematical model. 4. Regulation of cell flow in a transit population

The role of cells capable of formation of megakaryocytic colonies (CFUm) in regulation of thrombocytopoiesis was studied using a simulation model of megakaryocytopoiesis. The CFUm were shown to be the least differentiated cells involved in regulation of megakaryocytopoiesis in mice upon immune thrombocytopenia and after intravenous injection of hydroxyurea. A correlation was found between the CFUm population productivity and the number of cells in a transit population of the megakaryocytic line which makes it possible to reproduce experimental kinetics in the model. The duration of cell development from CFUm to megakaryocytes is about 3 days.     

A study of the effect of radiation on megakaryocytopoiesis using a mathematical model

Mathematical modelling used in analysing the postirradiation changes in megakaryocytopoiesis permitted to determine the level of radiation-induced injury in each experiment conducted and to show that megakaryocytopoiesis regulation followed the same mechanism after irradiation as it does normally and after the effect of hydroxyurea and anti-thrombocyte serum. The analysis has demonstrated that after the stem cell death induced by ionizing radiation, the regeneration can be provided by the committed cells, and the level of regeneration is determined by the maturity of precursors.     

Humoral regulation of megakaryocytopoiesis

If compared to erythroid and granulomacrophage lineages, the knowledge of the regulation of megakaryocytopoiesis has progressed slowly, and only the recent advent of specific clonogenic methods has permitted studies aimed at investigating this aspect of hematopoiesis. The analysis of Mk differentiation and platelet production is still difficult, because methods such as the 75SeM or 35S incorporation are time consuming and their sensitivity is relatively low. A number of laboratories have been able to purify, partially or to homogeneity, fractions stimulating the proliferation and differentiation of megakaryocytes. The biochemical identity between IL-3 and the active fractions found in the C.M. of some cell lines stands for a role of this hemopoietin in the regulation of megakaryocytopoiesis. However, the function of Epo and, above all, of GM-CSF cannot be ruled out, on the basis of experimental works, although only in some clinical trials GM-CSF seems to have been able to modify the platelet number. Hopefully, data on the therapeutic use of rhIL-3, and the sequentiation and identification of a molecule capable of action on the maturative compartment will shed new light on the regulation of megakaryocytopoiesis and the possibility to correct its disorders.     

Animal models with inherited hematopoietic abnormalities as tools to study thrombopoiesis

Animals with hereditary abnormalities of hematopoiesis are quite useful in the study of regulatory pathways of megakaryocytopoiesis and platelet formation. Seven such animal models are analyzed here. The Wistar Furth rat has been recently discovered to have reduced platelet number, but large mean platelet volume, and is, therefore, a model of hereditary macrothrombocytopenia. Study of the Wistar Furth rat may help to elucidate the process of platelet formation. Two mouse mutants the S1/S1d and W/Wv, have macrocytic anemia with reduced megakaryocyte number, but normal platelet count. In these mice, the platelet count is maintained by increased platelet production per megakaryocyte. These models demonstrate that factors other than platelet level are monitored in the feedback regulation of megakaryocytopoiesis and platelet production, and further study should lead to a better understanding of the regulation of megakaryocyte size. The Belgrade rat has severe microcytic anemia with decreased megakaryocyte number. Megakaryocyte size is increased, but platelet count is moderately reduced and thus the megakaryocyte-platelet picture resembles that of severe iron deficiency anemia. A more in depth examination of this model should delineate the effects of iron deficiency and hypoxia on megakaryocytopoiesis. The grey collie dog has cyclic hematopoiesis with large asynchronous fluctuations in all blood cell counts at approximately 2-week intervals. Megakaryocytes have not been studied. This model should be a tool to define the relationships between hematopoietic growth factors and differentiation of the various hematopoietic cell lineages. The br/br rabbit has a transient disturbance in fetal megakaryocytopoiesis and brachydactyly due to spontaneous amputation. Further study of this model may provide a better understanding of fetal megakaryocyte development and establish whether an association exists between the abnormal megakaryocytes and the limb amputations. The nude mouse with its severe T-lymphocyte deficiency has been studied to ascertain whether T cells play a regulatory role in normal and acute thrombocytopenia-stimulated megakaryocytopoiesis. The question of whether T cells or their products are responsible for reactive thrombocytosis in chronic inflammation could be examined with this model. These animal mutants have provided and should continue to provide important models for understanding the regulation of megakaryocytopoiesis and platelet production.     

A biological and computational model of megakaryocyte development as a stochastic branching process

The purpose of this paper is to describe a model of megakaryocytopoiesis as a branching process with stochastic processes regulating critical control points of differentiation along the stem cell megakaryocyte platelet axis. Progress of cells through these critical control points are regulated by transitional probabilities, which in turn are regulated by influences such as growth factors. The critical control points include transition of resting megakaryocytic stem cells (CFU-meg) into proliferating stem cells, the cessation of cytokinesis, and the cessation of DNA synthesis. A computerized computational method has been developed for directly fitting the stochastic branching model to colony growth data. The computational model has allowed transitional probabilities to be derived from colony size data. The model provides a unifying explanation for much of the heterogeneity of stages of maturation within populations of megakaryocytes and is fully compatible with historical data supporting the stochastic nature of hematopoietic stem cell regulation and with modern molecular concepts about control of the cell cycle.     

Megakaryocytopoiesis studied in a mathematical model. 2. Regulation of cell differentiation following damage to part of the proliferating bone marrow pool by hydroxyurea

Analysis of the earlier obtained data on the effect of hydroxyurea on megakaryocytopoiesis by a simulation model has shown that differentiation of proliferating precursors of the megakaryocytes depends on their amount in bone marrow; the time of their involvement in the proliferating pool increases if the number of proliferating cells decreases. Within 24 h after the treatment with hydroxyurea (900 mg/kg), the transit time for the mouse megakaryocytes is reduced by about 14 h due to acceleration of the latest developmental stages. A hypothesis is put forward which accounts for correlation between the duration of proliferative period and the volume and maturation time of megakaryocytes.     

Effect of erythroid differentiation factor on megakaryocytic differentiation of L8057, a murine megakaryoblastic leukemia cell line.

To assess the potent effect of erythroid differentiation factor (EDF) on megakaryocytopoiesis, effect of EDF on megakaryocytic differentiation of L8057, a murine megakaryoblastic cell line, was examined. EDF potentiated AchE induction of L8057 in a dose dependent manner. The potency of EDF on megakaryocytic differentiation is comparable to that on erythroid differentiation reported previously. The present results suggest that EDF may play a regulatory role in megakaryocytopoiesis as well as in erythropoiesis.     

Maturation and regulation of megakaryocytopoiesis.

The in vitro cloning technique for detecting megakaryocyte precursor cells was employed to compare stimuli known to influence megakaryocytopoiesis. Preparations of thrombopoietic stimulating factor (TSF) did not directly stimulate the growth of megakaryocyte colonies (CFU-m) but increased the frequency of CFU-m when TSF was added to the cultures with a constant amount of megakaryocyte colony stimulating factor. Platelets or platelet homogenates did not influence the frequency of CFU-m or the size of individual colonies. Analysis of cell surface properties of megakaryocytes obtained either by isolation from bone marrow or from in vitro colonies revealed species differences. The possibility that megakaryocytopoiesis and platelet release are regulated both within the marrow as well as by humoral factors is discussed.     

Manipulations of cholinesterase gene expression modulate murine megakaryocytopoiesis in vitro.

Megakaryocytopoiesis was selectively inhibited in cultured murine bone marrow cells by a 15-mer oligodeoxynucleotide complementary to the initiator AUG region in butyrylcholinesterase mRNA. Furthermore, conditioned medium from Xenopus oocytes producing recombinant butyrylcholinesterase stimulated megakaryocytopoiesis. These observations implicate butyrylcholinesterase in megakaryocytopoiesis and suggest application of oligodeoxynucleotides for modulating bone marrow development.     

Atypical micromegakaryocytes, promegakaryoblasts and megakaryoblasts: a critical evaluation by immunohistochemistry, cytochemistry and morphometry of bone marrow trephines in chronic myeloid leukemia and myelodysplastic syndromes.

A morphometric analysis of bone marrow trephine biopsies has been performed to study the frequency and planimetric characteristics of so-called atypical micromegakaryocytes in chronic myeloid leukemia (CML) and myelodysplastic syndromes (MDS). In addition, an attempt was made to discriminate this particular cell population from small immature elements of megakaryocytopoiesis, such as promegakaryoblasts and megakaryoblasts. The staining reactions employed included periodic acid-Schiff (PAS), alpha-naphthyl acetate esterase (ANAE) and immunohistochemistry with a monoclonal antibody against platelet glycoprotein IIIa (Y2/51-CD61). Comparison of the various staining reactions applied to the different megakaryocytic elements together with morphometric measurements resulted in a clearcut identification of promegakaryoblasts. These were defined as the earliest immature and exclusively CD61-positive precursors. Atypical micromegakaryocytes were characterized by their dysplastic features and strong ANAE reactivity in addition to their positive CD61 staining. When stringent diagnostic criteria (diameter ranging between 10 to 15 microns, mean size about 12 microns) were applied, this abnormal cell population comprised less than 10% of total megakaryocytopoiesis in CML and MDS. It may be assumed that dysmegakaryocytic features in the latter disorders are partially generated by small to medium-sized megakaryocytes (diameter less than 30 microns). In conclusion, the relative frequency of promegakaryoblasts in the normal bone marrow (range 6-8%) is confirmed by evaluation of the immunohistochemical and cytochemical staining methods (CD61 and ANAE). Furthermore, the ANAE reaction facilitates the recognition of atypical micromegakaryocytes as well as small megakaryocytes. Thus cytochemistry provides a better insight into alterations of these cell lineages in various pathological conditions.     

Thrombin-stimulated effects on megakaryocytopoiesis and pulmonary-platelet interactions

The effects of thrombin stimulation on megakaryocytopoiesis and pulmonary-platelet interactions were investigated before and after administration of the compound to 15 mongrel dogs. Each dog served as its own control. Thrombin was given to encourage the traffic of megakaryocytes into the lung and to study the thrombin-stimulated effects on megakaryocytopoiesis in the bone marrow. Our results showed that thrombin increased the numbers of bone marrow cells in general and megakaryocytes (MK) in particular. In addition, the maturation cycle of megakaryocytes was accelerated and the number of MK migrating into the central venous circulation was nearly doubled. Most of the circulating MK ultimately became sequestered in pulmonary capillaries, where platelets were shed into the arterial circulation. We conclude that thrombin has a major stimulatory effect on megakaryocytopoiesis in the bone marrow and that the lung plays an important role as a vascular filter and regulator of circulating platelet count.     

Ability of plasma from patients with immune thrombocytopenic purpura (ITP) to promote the growth of megakaryocyte progenitors from normal bone marrow.

The ability of plasma from ITP patients (before and after splenectomy) to support the growth of megakaryocyte progenitors was compared with that from healthy subjects. Plasma Factor Index-Megakaryocyte PFI-Mk (ITP) which expressed resultant colony growth was significantly lower before splenectomy, but it normalized after splenectomy. (PFI-Mk) (ITP) did not relate neither to megakaryocyte nor to platelet counts. A positive correlation has been observed between megakaryocyte and platelet numbers in healthy subjects and in ITP patients after splenectomy, but not before splenectomy. The proportion of immature megakaryocytes was markedly higher in ITP marrow before splenectomy. This study indicates, that in ITP apart from antibodies directed to platelets and megakarocytes a low plasma stimulatory activity affected megakaryocytopoiesis.     

Human megakaryocytopoiesis--normal and abnormal.

Regulation of megakaryocyte and platelet production remains poorly understood. In culture system two separate activities are needed for maximum production of megakaryocyte progenitors: promotor of clonal expansion and promoter of maturation, other growth factors and cells also contribute to regulation of megakaryocytopoiesis. Increased proliferation of megakaryocytes is observed in myeloproliferative disorders and idiopathic thrombocytopenic purpura, and decreased proliferation is found in aplastic anaemia and hypomegakaryocytic thrombocytopenia. Dysmegakaryocytopoiesis is present in myelodysplastic syndromes and acute leukaemia, and a proliferation of immature megakaryocytes in acute megakaryoblastic leukaemia. Increased understanding of human megakaryocytopoiesis is beginning to help in rational clinical management.     

New insights into the regulation of human megakaryocytopoiesis

It is apparent that multiple cellular stages and biologic processes can be identified during megakaryocytopoiesis that are potentially subject to control by hematopoietic growth factors and marrow accessory cell populations. Two classes of megakaryocyte progenitor cells, the colony forming unit-megakaryocyte (CFU-MK) and the burst forming unit-megakaryocyte (BFU-MK), have now been detected in normal human bone marrow cells. The BFU-MK by virtue of the greater cellular content of its resultant colonies and the delayed time of appearance of these colonies appears to be a more primitive progenitor cell with a greater proliferative potential than the CFU-MK. A number of hematopoietic growth factors including megakaryocyte colony stimulating factor, (MK-CSF), recombinant erythropoietin (EPO) and granulocyte macrophage colony stimulating factor (GM-CSF) are each capable of increasing cloning efficiency of human megakaryocyte progenitor cells. It is presently unknown whether these factors act directly on the CFU-MK or whether they stimulate marrow accessory cells to elaborate growth factors that influence CFU-MK proliferation. In order to answer this question, the effect of these growth factors on the cloning efficiency of a human megakaryocytic cell line, EST-IU, was examined. Each of these factors was capable of increasing leukemia cell colony formation. One can conclude from these studies that MK-CSF, EPO, and GM-CSF act directly on cells of the megakaryocytic lineage. The physiologic significance of the lineage nonspecific effects of EPO and GM-CSF on megakaryocytopoiesis is yet to be determined. On the basis of these observations, a model of human megakaryocytopoiesis was suggested. Several factors appear able to influence multiple steps in megakaryocytic development, whereas others influence only specific stages or cellular events occurring during megakaryocytopoiesis.     

Cyclic-AMP inhibits cell growth and negatively interacts with platelet membrane glycoprotein expression on the Dami human megakaryoblastic cell line

Intracellular signaling processes by which hematopoietic growth factors regulate megakaryocytopoiesis remain uncompletely understood. Cyclic AMP (cAMP) has been shown to be implicated in the regulation of growth and differentiation in various normal and malignant cell types. Since a few studies have suggested the possible involvement of the cAMP pathway as one of the intracellular mechanisms whereby megakaryocytopoiesis may be regulated, we investigated the functional effects of cAMP on the human megakaryoblastic Dami cell line. We observed that exposure of Dami cells to cAMP analogs or to agents elevating intracellular cAMP levels yielded dose-dependent cell growth inhibition. Cell cycle progression analysis of cells predominantly synchronized at the G1/S boundary by prior treatment with hydroxyurea revealed that cAMP transiently accumulated cells in the G2/M phase, then slowing down cell cycle. On the other hand, immunofluorescence and Northern blot analysis of megakaryocytic differentiation marker expression showed that probes we have used significantly inhibited GPlb expression. Moreover, although these agents used alone did not affect GPllb/llla expression, they markedly reversed phorbol ester-induced GPllb/llla expression increase. These inhibitory cAMP actions on glycoprotein expression were not the result of cell cycle perturbation since we observed that GPlb and GPllb/llla expression were not cell cycle dependent. All these data may then be consistent with a potential negative regulatory role of the cAMP intracellular signaling pathway during megakaryocytopoiesis. © 1995 Wiley-Liss, Inc.     

Induction of megakaryocyte differentiation by a novel pregnancy-specific hormone.

Maturation of megakaryocytes and subsequent platelet release are normally regulated by a network of cytokines, including thrombopoietin and various interleukins. Because abnormal platelet production and activation have been implicated in gestational pathologies, additional pregnancy-specific cytokines may play important roles in the regulation of megakaryocytopoiesis. Consistent with this hypothesis, we have found that the hormone prolactin-like protein E, a placental hormone that we have recently characterized, targets megakaryocytes through a specific cell surface receptor and induces megakaryocyte differentiation through a gp130-dependent signal transduction pathway.     

Mouse models of diseases of megakaryocyte and platelet homeostasis

Platelets are the small anuclear blood cells that are the product of megakaryocytopoiesis, the process of hematopoietic stem cell commitment to megakaryocyte production and the differentiation and maturation of these cells for platelet release. Deregulation or disruption of megakaryocytopoiesis can result in platelet deficiencies, the thrombocytopenias, with attendant risk of hemorrhage or thrombocytosis, a pathological excess of platelet numbers. Mouse models, particularly those engineered to carry genetic alterations modeling mutations associated with human disease, have provided important insights into megakaryocytopoiesis and deregulation of this process in disease. This review focuses on mouse models of diseases of altered megakaryocyte and platelet number, illustrating the profound contribution of these models in validating suspected roles of disease-associated genetic alterations, promoting discovery of new links between genetic mutations and specific diseases, and providing unique tools for better understanding of disease pathophysiology and progression, as well as resources to define drug action or develop new therapeutic strategies.     

Co-stimulatory effect of TLR2 and TLR4 stimulation on megakaryocytic development is mediated through PI3K/NF-ĸB and XBP-1 loop

Toll-like receptors (TLRs) are a class of proteins (patterns recognition receptors-PRRs) capable of recognizing molecules frequently found in pathogens (that are so-called pathogen-associated molecular patterns-PAMPs), they play a key role in the initiation of innate immune response by detecting PAMPs. Our findings show that the functional effects of TLRs co-stimulation on megakaryocytopoiesis. A single cell may receive multiple signal inputs and we consider that multiple TLRs are likely triggered during infection by multiple PAMPs that, in turn, might be involved in infection driven megakaryocytopoiesis, and the present study provide the evidence for the megakaryocytic effects of TLRs co-stimulation.     

Partial purification of human urinary megakaryocyte colony-stimulating factor

Urinary extracts from patients with aplastic anemia are known to promote murine megakaryocytopoiesis. In this report, we show a simple method for the partial purification of megakaryocyte colony-stimulating factor from human urine. A four-step purification procedure, which included ethanol precipitation, CM Affi-Gel Blue chromatography, wheat germ agglutinin-Sepharose chromatography and high-resolution hydroxyapatite chromatography, resulted in an about 430- to 630-fold increase of specific activity. The final fractions were still contaminated with erythropoietin, but the contaminated content of erythropoietin was not enough to stimulate mouse megakaryocytopoiesis in our culture system. We also demonstrate that human urinary extracts stimulated human megakaryocyte colony formation.